肾小球内皮细胞（GEnCs）损伤是糖尿病肾病（DKD）产生蛋白尿和肾小球硬化的主要原因之一，但机制尚未明了。我们的前期研究提示糖尿病导致的miR-124-ROCK1通路异常可能是DKD肾小球内皮细胞损伤、蛋白尿发生的重要机制。为证实该假设，本项目拟采用小鼠GEnCs、诱导型内皮细胞特异性表达miR-124的转基因小鼠为研究对象，通过细胞转染、原位杂交、SmartFlare RNA探针、肾小球分离等技术分别从分子、细胞和动物模型层面进一步阐述糖尿病引起GEnCs内miR-124降低，后者靶向上调ROCK1导致细胞损伤、继而导致DKD进展的新机制；并观察PPAR-γ激动剂吡格列酮是否通过调节miR-124-ROCK1通路改善DKD的早期病变和蛋白尿。本项目如获资助，将为调节miR-124-ROCK1通路作为防治DKD早期内皮损伤和蛋白尿的新靶点提供充分依据。

Nowadays, the pathogenesis, precaution and treatment of Diabetic kidney disease (DKD) are the major focus in Nephrology research. We previously reported that ROCK1 up-regulation and activation in glomerular endothelial cells (GEnCs) is one of the key mechanisms leading to DKD. Since up-regulation of ROCK1 protein is not associated with increase in its mRNA level, this indicates there should be an alternative mechanism to promote ROCK1 expression. In our preliminary study, we found that miR-124 could be an important factor that suppresses the translation of ROCK1. The evidence include: the levels of miR-124 in the glomeruli of DKD patients or diabetic mice are significantly decreased compared the results in normal controls. Moreover, high glucose significantly suppresses miR-124 expression in GEnCs. These results suggest that down-regulation of miR-124 and hence increase in ROCK1 translation, might be a crucial mechanism in the development of DKD. To prove this hypothesis, we propose below specific aims: 1) in vitro, we plan to investigate if down-regulation of miR-124 promotes ROCK1 expression in GEnCs after stimulated with high glucose. To this end, we will use several molecular techniques including miRNA mimic transfection, dual luciferase reporter assay. 2) in vivo, we will employ loxP-Cre technique to generate tamoxifen-inducible, endothelial-specific miR-124 overexpression mice, and then to examine if these mice would be resistant to develop DKD in diabetic condition induced by STZ. We will use situ hybridization, SmartFlare RNA technique and histological analysis in this study. 3) by pharmacological targeting miR-124, we will explore if pioglitazone treatment could enhance miR-124 to suppress ROCK1 in db/db mice with DKD. If our project were granted, it should provide sufficient scientific evidence to identify miR124-ROCK1 pathway as a new target to prevent early GEnCs injury in DKD.